I. Field of the Invention
The present invention relates generally to the field of treating cells with transient electric fields and more particularly to electroporation of cells. More particularly, it concerns methods for regulated flow electroporation, an electroporation chamber and related devices.
II. Description of Related Art
Electroporation has been used for the insertion of molecules into animal or plant cells since the early 1970's. Researchers have demonstrated that exposure of cells to a short duration, high voltage electrical field causes openings to form in the cell membrane through which molecules, including macromolecules such as proteins and DNA, can enter the cell. These openings, referred to as electropores, are regions of increased permeability initiated by local breakdown in the cell membrane caused by high voltage electric fields. These pores exist transiently but long enough for macromolecules such as plasmid DNA molecules to enter the cells. While cells can tolerate the creation of these pores, the process of creating them and introducing molecules can, if carried out to excess, kill the cells. Since this electroporation involves passing a current (albeit briefly) through the conductive cell suspension the electrodes generate heat in accordance with Joule's Law. This heat can increase the temperature of the cell suspension and, if excessive, raise the temperature of the cells to a point where the cells die.
Most early applications of electroporation were carried out using specialized cuvettes containing electrodes positioned relative to one another so that a substantially uniform electric field can be generated between them. Most conveniently, this is provided by two flat plate electrodes attached to opposite walls of a rectangular cuvette or chamber. A suspension of cells to be electroporated combined with a molecule or molecules that the operator desired to introduce into the cells is placed in the cuvette or chamber, which is then placed between the electrodes such that the cell suspension comes into in fluid contact with the electrodes. To effect electrophorectic introduction of the molecules into the cells, an electric field pulse of high voltage and short duration was applied one or more times to the electrodes and thereby to the cell suspension between the electrodes. Most commercially available electroporation cuvettes are limited in capacity and can only process small amounts of cell suspension at a time (usually less than one ml). Given the small volumes of cells that could be electroporated per loading of a cuvette, electroporation of large volumes of cell suspension was impractical.
Typically, maintenance of sterility is essential for nearly all applications of electroporation of large volumes of cells. Relative to the maintenance of sterility, repeated loading of a cuvette and pooling of the electroporated cells is especially impractical. While this method of electroporation is convenient and simple and met the needs of many researchers carrying out small scale electroporation of cells additional methods were needed, especially methods that could conveniently facilitate the electroporation of large volumes of cells while maintaining a sterile environment. Electroporation of large volumes of cells in a closed sterile system would enable the use of electroporation for cell based therapy of humans. A method and apparatus that creates and maintains a closed sterile fluid path throughout the operation is therefore most desired.
In the 1980's, work was initiated by several researchers on flow electroporation for processing large volumes of cells (U.S. Pat. Nos. 4,752,586, 5,612,207, 6,074,605, 6,090,617, each of which is incorporated herein in its entirety by reference). Flow devices for electroporation generally consisted of parallel electrodes between which the cell suspension to be electroporated continuously and steadily flowed until the entire volume of cells had been electroporated. As the cell suspension steadily flowed between the electrodes, a high voltage pulse was applied to the cells. Repeated application of high voltage pulses to the electrodes resulted in the generation of heat and it was found to be necessary to remove this heat by a cooling means to prevent the electrodes and the cell suspension from reaching too high a temperature. The apparatus for flow electroporation contain an electroporation chamber having the electrodes and the ports through which cell suspension can be pumped. The electrodes are connected electrically to electronic circuitry that is capable of providing high voltage pulses to the electrodes. The high voltage pulses can be controlled by a programmable computer. Flow electroporation systems have been developed that are capable of electroporating larger volumes of cells, enabling production of viable cells into which a desired molecule had been introduced.
The conditions for electroporation vary from cell type to cell type and can vary according to the type of molecule one desires to introduce into the cells. For any particular cell type an optimal process exists consisting of an optimal number pulses of optimal voltage and duration spaced at optimal intervals. In order to apply the optimal number of electrical pulses to the cells, when using a flow electroporation apparatus described in the art, the rate of flow of the cell suspension through the electroporation chamber and between the electrodes, as well as the rate of pulsing is chosen to provide the optimal number of pulses to a volume of cell suspension. For example, if the optimal number of pulses per cell is known to be two per unit time for a particular cell, and the volume through the electroporation chamber is one milliliter, then the flow rate is set to one milliliter per unit time and the two pulses are applied per unit time. In this way on average, each cell will receive two pulses in this example. However, the hydrodynamic flow of the cell suspension through the electroporation chamber does not result in every cell traveling through the chamber and between the plates at the same rate. Since the rate of flow is higher away from the chamber walls than near the chamber walls a cell that flows between the electrodes toward the center of the fluid flow will pass between the electrodes in less than the unit time, and may receive fewer than two pulses, while a cell that flows near a wall may take longer than the unit time to pass between the electrodes and thereby receive more than two pulses. Since in this example two pulses are optimal for every cell, clearly not every cell receives the optimal number of pulses and the overall electroporation of the cell suspension is less than optimal.
As mentioned above, electroporation inherently results in the production of heat in the cell suspension, according to Joule's Law. Since in a flow electroporation method in which flow is steady and continuous, heating is substantially continuous the means to remove heat from the electroporation chamber must be capable of balancing the production of heat to avoid having the temperature of the chamber rise to an unacceptable temperature. The continuous nature of the process does not provide for periods during which cooling can be provided between periods of electroporation.
As discussed above the optimal conditions for electroporation are likely to vary according to the particular cell type being electroporated and the type of molecule one desires to introduce into the cell by electroporation. It is possible to experimentally address this question by systematically varying electroporation conditions using static cuvettes and then apply the optimal conditions determined from these experiments to a flow electroporation system. This approach suffers from two drawbacks. First, since static cuvette electroporation of a large number samples is time consuming the cells used for each static electroporation will vary through the experiment. Second, using this approach one assumes, but does not know, that the optimal conditions for static cuvette electroporation will be the same as flow electroporation. It would be desirable to be able to optimize electroporation conditions rapidly using a single sample of cells for all experimental electroporation conditions while also using the same apparatus that later would be used for large-scale electroporation of cells for therapeutic or other applications.
Certain molecules, most notably mRNA, may be unstable in the presence of cells into which one desires to introduce the molecule by electroporation. In the case of mRNA, this is likely to be the result of ribonucleases present in the cell culture media or on the surface of the cells. In such cases, it is desirable to minimize the time during which the molecule will be in this unstable condition prior to its introduction into the cell. With static cuvette based electroporation the molecule (e.g., mRNA) is mixed with the cells and then manually loaded into the cuvette, whereupon following installation in the electroporation apparatus electroporation is carried out. During the time required to carry out these manual steps some of the molecules added to the cells may be destroyed. A method whereby the molecules to be introduce can be automatically mixed with cells immediately prior to electroporation and in a closed sterile environment is desirable.
In a continuous flow system described in the art, apart from coordinating the application of pulses to the electrodes with the initiation of flow of cells between the electrodes there is no “during process” coordination between pulsing and flow of the cells.
A method and apparatus that combines the advantages of continuous flow electroporation, particularly the ability to electroporate large volumes of cells in a sterile closed system while assuring that every cell receives the optimal number of pulses, is desirable.